Surface-assisted laser desorption/ionization method, mass spectrometry method and mass spectrometry device

ABSTRACT

A surface-assisted laser desorption/ionization method according to an aspect includes: a first process of preparing a sample support having a substrate in which a plurality of through-holes passing from one surface thereof to the other surface thereof are provided and a conductive layer that covers at least the one surface; a second process of placing a sample on a sample stage and arranging the sample support on the sample such that the other surface faces the sample; and a third process of applying a laser beam to the one surface and ionizing the sample moved from the other surface side to the one surface side via the through-holes due to a capillary phenomenon.

TECHNICAL FIELD

The present invention relates to a surface-assisted laserdesorption/ionization method, a mass spectrometry method and a massspectrometry device.

BACKGROUND ART

As a technique for ionizing a sample such as a biological sample inorder to perform mass spectrometry or the like, matrix-assisted laserdesorption/ionization (MALDI) has been known thus far. MALDI is atechnique for ionizing a sample by mixing the sample with alow-molecular weight organic compound, called a matrix, absorbing anultraviolet laser beam, and applying the laser beam to the mixture.According to this technique, a heat-labile substance or a high-molecularweight substance can be subjected to non-destructive ionization(so-called soft ionization). However, MALDI generates background noisederived from the matrix.

As a technique for performing ionization without using such a matrix,surface-assisted laser desorption/ionization (SALDI) for ionizing asample by using a substrate whose surface has an uneven microstructureis known. For example, as an ionization method of a sample according toSALDI, there is a method of using a surface having anodized porousalumina, anodized porous silicon, or the like having fine concavities asa sample holding surface (see Patent Literatures 1 and 2 below). In thisionization method, a sample to be analyzed is dropped onto the sampleholding surface having the fine concavities, and a laser beam is appliedafter drying the sample to ionize the sample.

CITATION LIST Patent Literature

-   [Patent Literature 1] Japanese Patent No. 5129628-   [Patent Literature 2] U.S. Pat. No. 6,288,390

SUMMARY OF INVENTION Technical Problem

However, in the above ionization method, since a positional deviation ofthe sample with respect to the substrate occurs when dropping thesample, it is difficult to ionize the sample while maintaining originalposition information of the sample (a two-dimensional distribution ofmolecules composing the sample). For this reason, it is difficult tomeasure what kind of and how many molecules are present at each positionof a sample region and use the ionization method in imaging massspectrometry or the like imaging a two-dimensional distribution map ofthe sample molecules. Even when a method of transferring the sample tothe substrate instead of dropping the sample onto the substrate isadopted, there is a problem in that a positional deviation of the samplewith respect to the substrate occurs when transferring the sample or anuneven transfer of the sample occurs.

Therefore, an aspect of the present invention is directed to providing asurface-assisted laser desorption/ionization method capable of ionizinga sample while maintaining positional information of the sample, a massspectrometry method, and a mass spectrometry device.

Solution to Problem

A surface-assisted laser desorption/ionization method according to anaspect of the present invention includes: a first process of preparing asample support having a substrate in which a plurality of through-holespassing from one surface thereof to the other surface thereof areprovided and a conductive layer that is formed of a conductive materialand covers at least the one surface; a second process of placing asample on a sample stage and arranging the sample support on the samplesuch that the other surface faces the sample; and a third process ofapplying a laser beam to the one surface and ionizing a sample movedfrom the other surface side to the one surface side via thethrough-holes due to a capillary phenomenon.

According to the surface-assisted laser desorption/ionization method,the substrate in which the plurality of through-holes are provided isarranged on the sample, and thereby the sample can be raised from theother surface side toward the one surface side of the substrate via thethrough-holes due to the capillary phenomenon. Thereby, the sample canbe moved from the other surface side to the one surface side of thesubstrate while positional information of the sample (two-dimensionaldistribution of molecules composing the sample) is maintained. The laserbeam is applied to the one surface of the substrate, and energy thereofis transmitted to a sample moved to the one surface side via theconductive layer. Thereby, the sample is ionized. As a result, thesample can be ionized while the positional information of the sample ismaintained. Therefore, according to the aforementioned method, thesample can be ionized by a simple operation in which the substrate inwhich the plurality of through-holes are provided is placed on thesample while the positional information of the sample is maintained.

The substrate may be formed by anodizing a valve metal or silicon. Thevalve metal or silicon is anodized, and thereby the sample supporthaving the substrate in which the plurality of through-holes areprovided is used. Thereby, the movement of the sample caused by theaforementioned capillary phenomenon can be properly realized.

Each of the through-holes may have a width of 1 to 700 nm. The substratehaving the through-holes each having the hole width of 1 to 700 nm isused, and thereby the movement of the sample caused by theaforementioned capillary phenomenon can be more smoothly performed.Sufficient signal intensity can be obtained in mass spectrometry usingthe surface-assisted laser desorption/ionization method.

The substrate may have a thickness of 5 to 10 μm. Thereby, strength ofthe substrate can be ensured, and sufficient signal intensity can beobtained in mass spectrometry using the surface-assisted laserdesorption/ionization method.

The sample support may further include a frame mounted on an outer edgeof the one surface of the substrate. Bending of the substrate issuppressed by the frame, and the sample support is easily handled whensupported or moved. As a result, an arrangement of the sample support onthe sample in the second process can be easily performed.

In the second process, the sample support may be fixed to the samplestage. As the sample support is fixed to the sample stage, the sampleand the sample support are brought into close contact with each other,and movement of the sample caused by the capillary phenomenon can besmoothly performed. Sideslippage of the sample support arranged on thesample can be prevented, and a positional deviation of the sample causedby the sideslippage of the sample support can be suppressed.

A surface-assisted laser desorption/ionization method according toanother aspect of the present invention includes: a first process ofpreparing a sample support having a substrate which is formed of aconductive material and in which a plurality of through-holes passingfrom one surface thereof to the other surface thereof are provided; asecond process of placing a sample on a sample stage and arranging thesample support on the sample such that the other surface is in contactwith the sample; and a third process of applying a laser beam to the onesurface and ionizing a sample moved from the other surface side to theone surface side via the through-holes due to a capillary phenomenon.

In the surface-assisted laser desorption/ionization method, thesubstrate formed of a conductive material is used. Thereby, theconductive layer can be omitted, and the same effects as when the samplesupport having the aforementioned conductive layer is used can beobtained.

A mass spectrometry method according to an aspect of the presentinvention includes: each of the processes of the surface-assisted laserdesorption/ionization method; and a fourth process of detecting thesample ionized in the third process, wherein the application of thelaser beam in the third process and the detection of the ionized samplein the fourth process are performed at each application position whilechanging application positions of the laser beam.

According to the mass spectrometry method, the sample can be ionized bya simple operation in which the sample support is arranged on the samplewhile positional information of the sample is maintained. While changingthe application positions of the laser beam, the ionized sample isdetected at each application position, and thereby two-dimensionaldistribution of sample molecules can be perceived. Therefore, accordingto the mass spectrometry method, imaging mass spectrometry for imaging atwo-dimensional distribution map of the sample molecules can beperformed by a simple operation.

A mass spectrometry device according to an aspect of the presentinvention includes: a sample stage on which a sample is placed; a laserbeam application unit configured to, in a state in which a samplesupport, which has a substrate in which a plurality of through-holespassing from one surface thereof to the other surface thereof areprovided and a conductive layer that is formed of a conductive materialand covers at least the one surface, is arranged on the sample placed onthe sample stage such that the other surface faces the sample, apply alaser beam to the one surface while changing application positionsthereof; and a detection unit configured to detect a sample ionized bythe application of the laser beam at each of the application positions.

According to the mass spectrometry device, the sample can be ionized bya simple operation in which the sample support is arranged on the samplewhile positional information of the sample is maintained. The laser beamapplication unit applies the laser beam while changing the applicationpositions of the laser beam, and the detection unit detects the ionizedsample at each application position. Thereby, two-dimensionaldistribution of sample molecules can be perceived. Therefore, accordingto the mass spectrometry device, imaging mass spectrometry for imaging atwo-dimensional distribution map of the sample molecules can beperformed by a simple operation.

A mass spectrometry device according to another aspect of the presentinvention includes: a sample stage on which a sample is placed; a laserbeam application unit configured to, in a state in which a samplesupport, which has a substrate which is formed of a conductive materialand in which a plurality of through-holes passing from one surfacethereof to the other surface thereof are provided, is arranged on thesample placed on the sample stage such that the other surface is incontact with the sample, apply a laser beam to the one surface whilechanging application positions thereof; and a detection unit configuredto detect a sample ionized by the application of the laser beam at eachof the application positions.

According to the mass spectrometry device, the substrate formed of aconductive material is used. Thereby, the conductive layer can beomitted, and the same effects as when the sample support having theaforementioned conductive layer is used can be obtained.

Advantageous Effects of Invention

According to the present invention, a surface-assisted laserdesorption/ionization method capable of ionizing a sample whilemaintaining positional information of the sample, a mass spectrometrymethod, and a mass spectrometry device can be provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating an outline of a mass spectrometrymethod according to an embodiment of the present invention.

FIG. 2 is a perspective view of a sample support used in the massspectrometry method according to the present embodiment.

FIG. 3 is a sectional view taken along line III-III of FIG. 2.

FIG. 4 is an enlarged plan view on an effective region R of the samplesupport of FIG. 2.

FIG. 5 is an enlarged sectional view of major parts of the samplesupport of FIG. 2.

FIG. 6 is view illustrating a process of manufacturing a substrate ofFIG. 2.

FIG. 7 is a view illustrating a procedure of the mass spectrometrymethod according to the present embodiment.

FIG. 8 is a view illustrating a procedure of the mass spectrometrymethod according to the present embodiment.

FIG. 9 is a view illustrating a procedure of the mass spectrometrymethod according to the present embodiment.

FIG. 10 is a view illustrating a relation between a hole width of athrough-hole and a mass spectrum.

FIG. 11 is a view illustrating a relation between the hole width of thethrough-hole and the mass spectrum.

FIG. 12 is a view illustrating a relation between the hole width of thethrough-hole and the mass spectrum.

FIG. 13 is a view illustrating a relation between the hole width of thethrough-hole and the mass spectrum.

FIG. 14 is a view illustrating a relation between a thickness of asubstrate and signal intensity.

FIG. 15 is a view illustrating a first modification of the samplesupport.

FIG. 16 is a view illustrating a second modification of the samplesupport.

FIG. 17 is a view illustrating a third modification of the samplesupport.

FIG. 18 is a view illustrating a mass spectrum according to massspectrometry using a sample support before being baked and a massspectrum according to mass spectrometry using a sample support afterbeing baked.

DESCRIPTION OF EMBODIMENTS

Hereinafter, preferred embodiments of the present invention will bedescribed in detail with reference to the drawings. Note that the sameor equivalent portions are denoted by the same reference signs in eachof the drawings, and duplicate descriptions thereof will be omitted.Dimensions of each member (or region) illustrated in the drawings or aratio of the dimensions may be different from actual dimensions or aratio of the actual dimensions in order to facilitate an understandingof the description.

An outline of a mass spectrometry method (including a surface-assistedlaser desorption/ionization (SALDI) method) according to the presentembodiment will be described using FIG. 1. As illustrated in (a) of FIG.1, in the mass spectrometry method, first, one sample 10 to be subjectedto mass spectrometry is placed on a sample stage 1. Further, a samplesupport 2 having a substrate in which a plurality of through-holes areprovided is arranged on the sample 10. Here, the sample 10 to besubjected to spectrometry is a thin film-like biological sample (ahydrous sample) such as a tissue section.

Subsequently, as illustrated in (b) of FIG. 1, the sample 10 is movedfrom a lower surface side of the sample support 2 to an upper surfaceside of the sample support 2 via the through-holes by a capillaryphenomenon. The sample 10 stays on the upper surface side of the samplesupport 2 due to surface tension.

Subsequently, as illustrated in (c) of FIG. 1, an ultraviolet laser beamis applied to the upper surface side of the sample support 2, andthereby the sample 10 moved to the upper surface side of the samplesupport 2 is ionized and emitted into a vacuum. To be specific, energyof the ultraviolet laser beam is transmitted from the sample support 2absorbing the energy to the sample 10 moved to the upper surface side ofthe sample support 2. The sample 10 obtaining the energy is evaporatedand obtains electric charges to be sample ions (an ionized sample) 11.The sample ions 11 emitted into the air in this way are detected by adetector 3, and the detected sample ions 11 are measured. In this way,mass spectrometry of the sample 10 is performed.

The mass spectrometry method according to the present embodiment usestime-of-flight mass spectrometry (TOF-MS) by way of example. An outlineof TOF-MS is shown below. In TOF-MS, a ground electrode (not shown) isprovided between the sample support 2 and the detector 3, and apredetermined voltage is applied to the sample support 2. Thereby, apotential difference occurs between the sample support 2 and the groundelectrode, and the sample ions 11 generated at the upper surface side ofthe sample support 2 are accelerated and moved toward the groundelectrode by the potential difference. Afterward, the sample ions 11 flyin a drift space in which there are no electric and magnetic fieldsprovided from the ground electrode to the detector 3, and finally reachthe detector 3. Here, since the potential difference between the samplesupport 2 and the ground electrode is constant with respect to any ofthe sample ions 11, energy given to each of the sample ions 11 isconstant. For this reason, the sample ions 11 having a smaller molecularweight fly in the drift space at a higher speed and reach the detector 3within a shorter time. In TOF-MS, mass spectrometry is performed on thebasis of differences in arrival time of the sample ions 11 at thedetector 3.

Next, the sample support 2 will be described using FIGS. 2 to 5. FIG. 2is a perspective view illustrating an external appearance of the samplesupport 2 (a substrate 21 and a frame 22). In practice, a plurality ofthrough-holes S are provided in the substrate 21, and the sample support2 is provided with a bonding layer G that bonds the substrate 21 and theframe 22, and a conductive layer 23 that covers surfaces of thesubstrate 21 and the frame 22 (including inner surfaces of thethrough-holes S). However, since these layers are extremely small withrespect to the substrate 21 and the frame 22, these layers are notillustrated in FIG. 2. Meanwhile, in FIG. 3, which is a sectional viewtaken along line III-III of FIG. 2, the through-holes S, the conductivelayer 23, and the bonding layer G are shown in dimensions larger thanactual dimensions in order to describe arrangement configurations of thethrough-holes S, the conductive layer 23, and the bonding layer G.

As illustrated in FIGS. 2 and 3, the sample support 2 is a samplesupport for the SALDI method and has the rectangular plate-likesubstrate 21 in which the plurality of through-holes S are provided topass from one surface 21 a thereof to the other surface 21 b thereof;and the frame 22 that is mounted on an outer edge of the one surface 21a of the substrate 21.

The one surface 21 a and the other surface 21 b of the substrate 21have, for instance, square shapes in which a length D1 of one sidethereof is 1 cm. A thickness d1 from the one surface 21 a to the othersurface 21 b of the substrate 21 is 1 to 50 μm. In the presentembodiment, the substrate 21 is formed of an insulating material by wayof example. The substrate 21 is, for instance, an alumina porous film inwhich the plurality of through-holes S, each of which has a nearlyconstant hole diameter, are formed by anodizing aluminum (Al). Thesubstrate 21 may be formed by anodizing a valve metal other than Al suchas tantalum (Ta), niobium (Nb), titanium (Ti), hafnium (Hf), zirconium(Zr), zinc (Zn), tungsten (W), bismuth (Bi), antimony (Sb), or the like,or by anodizing silicon (Si).

The frame 22 is provided along the outer edge of the one surface 21 a ofthe substrate 21 in a quadrilateral ring shape. A width D2 of the frame22 is, for instance, 2 mm. A thickness d2 of the frame 22 is, forinstance, 1.0 to 500 μm. An effective region R of the one surface 21 aof the substrate 21 which is not covered with the frame 22 is a squareregion of 0.6 mm squared. The effective region R functions as a regionfor moving the sample 10 from the other surface 21 b to the one surface21 a due to a capillary phenomenon (to be described below). The frame 22is provided at an outer edge of the substrate 21, and thereby bending ofthe substrate 21 is suppressed. Since a portion at which the frame 22 isprovided can be fixed or grasped, handling thereof is facilitated whenthe sample support 2 is supported or moved. In the present embodiment,the frame 22 is provided in the quadrilateral ring shape, but it may beprovided along the outer edge of the substrate 21 in an annular shape.The frame 22 is provided in the annular shape, and thereby the bendingof the substrate 21 is further suppressed than in a case in which theframe 22 is provided in the quadrilateral ring shape.

As illustrated in FIG. 3, the frame 22 is bonded to a surface (the onesurface 21 a) of the substrate 21 via the bonding layer G. As a materialof the bonding layer G, a bonding material emitting a small amount ofgas, such as low-melting-point glass, or an adhesive for a vacuum can beused. In the present embodiment, the frame 22 is bonded to the substrate21 by overlapping a portion in which the through-holes S are provided inthe one surface 21 a of the substrate 21 by way of example. For thisreason, the through-holes S allow bending of interfaces between theportion at which the frame 22 is provided and a portion at which theframe 22 is not provided in the substrate 21. Thereby, the substrate 21is inhibited from being broken on the boundary surface.

The frame 22 has nearly the same coefficient of thermal expansion as thesubstrate 21. The frame 22 is, for instance, a ceramic member or thelike having the same composition as the substrate 21. The frame 22 isformed of, for instance, glass or a metal. In this way, the coefficientsof thermal expansion of the substrate 21 and the frame 22 approximateeach other, and thereby deformation or the like (for instance, strainsof the substrate 21 and the frame 22 during thermal expansion) caused bya change in temperature can be prevented.

As illustrated in FIGS. 3 and 5, the sample support 2 has the conductivelayer 23 that covers the one surface 21 a and the other surface 21 b ofthe substrate 21, the inner surfaces of the through-holes S, and asurface of the frame 22. The conductive layer 23 is a layer formed of aconductive material provided to give conductivity to the insulatingsubstrate 21. However, the conductive layer 23 is not hindered frombeing provided even when the substrate 21 is formed of a conductivematerial. As the material of the conductive layer 23, a metal having lowaffinity (reactivity) with the sample 10 and high conductivity ispreferred due to reasons that will be mentioned below.

For example, when the conductive layer 23 is formed of a metal such ascopper (Cu) having high affinity with the sample 10 such as a protein,the sample 10 may be ionized with Cu atoms attached to sample moleculesin a process (to be described below) of ionizing the sample 10. That is,when a molecular weight of the sample ions 11 detected by the detector 3is measured, the measured weight deviates from an actual molecularweight of the sample 10 by a mass of the attached Cu, and hence accuratemeasurement is not performed. Therefore, as the material of theconductive layer 23, a metal having low affinity with the sample 10 ispreferred.

Meanwhile, a metal having high conductivity can give a constant voltagein an easy and stable way. For this reason, when a metal having highconductivity is used as the conductive layer 23, a constant voltage iseasily applied to the substrate 21 in order to generate a constantpotential difference between the aforementioned ground electrode and thesubstrate 21. In addition, since a metal having higher conductivityshows a tendency to have higher thermal conductivity, the energy of thelaser beam applied to the substrate 21 can be efficiently transmitted tothe sample 10 via the conductive layer 23. Therefore, as the material ofthe conductive layer 23, a metal having high conductivity is preferred.

From the above viewpoint, for example, gold (Au), platinum (Pt), or thelike is used as the material of the conductive layer 23. For example,the conductive layer 23 can be formed by forming a film of Au or Pt onthe one surface 21 a and the other surface 21 b of the substrate 21, theinner surfaces of the through-holes S, and the surface of the frame 22using a plating method, an atomic layer deposition (ALD) method, a vapordeposition method, a sputtering method, or the like. In addition to Auand Pt, for example, chromium (Cr), nickel (Ni), titanium (Ti), etc. canbe used as the material of the conductive layer 23.

FIG. 4 is an enlarged plan view of the effective region R of the samplesupport 2. In FIG. 4, black portions denote the through-holes S, andwhite portions denote partition wall portions at which the through-holesS are not formed. As illustrated in FIG. 4, the plurality ofthrough-holes S having approximately constant sizes are formed on thesurface of the substrate 21. The plurality of through-holes S may beforming at such a size that the sample 10 can be moved (raised) from theother surface 21 b to the one surface 21 a by a capillary phenomenon (tobe described below). As in the example of FIG. 4, the sizes of thethrough-holes S may be uneven, and portions at which the plurality ofthrough-holes S are coupled to one another may be present. An apertureratio of the through-holes S (an area of portions at which thethrough-holes S are formed/a whole area) in the effective region Rranges from 10% to 80% from a practical point of view, and particularlypreferably ranges from 60% to 80%.

As illustrated in FIG. 5, the through-holes S extend from the onesurface 21 a side to the other surface 21 b side of the substrate 21. Awidth d3 of each of the through-holes S is 1 to 700 nm. A thickness d4of the conductive layer 23 is, for instance, about 1 to 25 nm. Here, thewidth d3 of each of the through-holes S is a hole width after theconductive layer 23 is formed in the through-holes S. When the substrate21 having the through-holes S, each of which has a hole width of 1 to700 nm, is used, the movement of the sample 10 caused by theaforementioned capillary phenomenon can be more smoothly performed. Asin the present embodiment, when a sectional shape of each of thethrough-holes S is a nearly circular shape, the width d3 of each of thethrough-holes S refers to a diameter of each hole. Meanwhile, when thesectional shape of each of the through-holes S is not a circular shape,the width of each of the through-holes S refers to a diameter (aneffective diameter) of an imaginary cylinder fitted into each of thethrough-holes S.

Next, a process of manufacturing the sample support 2 will be describedusing FIGS. 3 and 6. First, a process of manufacturing the substrate 21will be described using FIG. 6. As illustrated in (a) of FIG. 6, an Al(Aluminum) substrate 50 that will become a material of the substrate 21is prepared. Subsequently, as illustrated in (b) of FIG. 6, the Alsubstrate 50 is anodized. Thereby, the Al substrate 50 is oxidized froma surface thereof, and an anodized film 51 having a plurality ofconcavities 51 a is formed. Subsequently, as illustrated in (c) of FIG.6, the anodized film 51 is peeled from the Al substrate 50, and a bottom51 b of the anodized film 51 is removed or perforated. Thereby, thesubstrate 21 in which the plurality of through-holes S passing from onesurface 21 a to the other surface 21 b thereof are provided is obtained.

After the substrate 21 is manufactured in this way, the frame 22 ismounted on an outer edge of the substrate 21 via the bonding layer Gsuch as low-melting-point glass or an adhesive for a vacuum. Thereby,the thing which is in a state before the conductive layer 23 is formedin the sample support 2 illustrated in FIG. 3 is obtained. Finally, theconductive layer 23 formed of Au or Pt is provided to cover the onesurface 21 a and the other surface 21 b of the substrate 21, the innersurfaces of the through-holes S, and the surface of the frame 22. Asdescribed above, the conductive layer 23 is formed by forming a film ofAu or Pt on the one surface 21 a and the other surface 21 b of thesubstrate 21, the inner surfaces of the through-holes S, and the surfaceof the frame 22 using a plating method, an ALD method, or the like.Thereby, the sample support 2 illustrated in FIG. 3 is manufactured.

In the anodization of Al, the substrate 21 is adjusted to have thethickness d1 of 1 to 50 μm, and each of the through-holes S is adjustedto have the width d3 of 1 to 700 nm. To be specific, a thickness of theAl substrate 50 prepared first or conditions such as a temperature, avoltage, etc. in the anodization of the Al substrate 50 are properlyset, and thereby the thickness d1 of the substrate 21 and the width d3of each of the through-holes S are formed to have predetermined sizes(sizes included in the above range).

Next, a procedure of the mass spectrometry method using the samplesupport 2 will be described using FIGS. 7 to 9. In FIGS. 7 to 9, theconductive layer 23, the through-holes S, and the bonding layer G arenot illustrated.

First, a mass spectrometry device 100 for performing mass spectrometryusing the sample support 2 will be described using FIG. 9. The massspectrometry device 100 comprises the sample stage 1 on which the sample10 is placed, a laser beam application unit 4, and the detector (thedetection unit) 3.

In a state in which the sample support 2 is arranged on the sample 10placed on the sample stage 1, the laser beam application unit 4 appliesa laser beam L to the one surface 21 a while changing applicationpositions thereof. Here, the sample support 2 is placed on the sample 10such that the other surface 21 b comes into contact with the sample 10via the conductive layer 23. The laser beam L applied by the laser beamapplication unit 4 is, for instance, an ultraviolet laser beam such as anitrogen laser beam (an N₂ laser beam) having a wavelength of 337 nm orthe like.

The detector 3 detects the sample 10 (the sample ions 11), which isionized by the laser beam L being applied from the laser beamapplication unit 4 at each application position. To be specific, thelaser beam application unit 4 performs two-dimensional scanning on theeffective region R of the sample support 2 according to a predeterminedmovement width and a predetermined moving direction, and applies thelaser beam L at each scanning position. The detector 3 detects thesample ions 11 generated by the laser beam L being applied at eachscanning position. Thereby, mass spectrometry can be performed at eachposition on the effective region R. Results of the mass spectrometry ateach position of the sample 10 obtained in this way are synthesized, andthereby imaging mass spectrometry for imaging a two-dimensionaldistribution map of sample molecules can be performed. A procedure ofthe mass spectrometry performed by the mass spectrometry device 100 willbe described below in detail using FIGS. 7 to 9.

First, the aforementioned sample support 2 is prepared (a firstprocess). The sample support 2 may be prepared by a person who performsthe mass spectrometry and manufactures the sample support 2 in personusing the mass spectrometry device 100, or by acquiring the samplesupport 2 from a manufacturer, a seller, or the like of the samplesupport 2.

Subsequently, as illustrated in (a) of FIG. 7, the sample 10 to besubjected to mass spectrometry is placed on a placement surface 1 a ofthe sample stage 1 and, as illustrated in (b) of FIG. 7, the samplesupport 2 is arranged on the sample 10 such that the other surface 21 bcomes into contact with the sample 10 via the conductive layer 23 (seeFIG. 3) (a second process). Here, to move the sample 10 targeted on thespectrometry to the one surface 21 a side of the substrate 21 accordingto a capillary phenomenon, the sample support 2 is arranged on thesample 10 such that the sample 10 is included within the effectiveregion R in the planar view. To smooth the movement of the sample 10caused by the capillary phenomenon (to be described below), a solution(for instance, an acetonitrile mixture or the like) for reducingviscosity of the sample 10 may be mixed with the sample 10.

Subsequently, as illustrated in (a) of FIG. 8, the sample support 2 isfixed to the sample stage 1 (a continuation of the second process).Here, as an example, four sides of the sample support 2 (upper andlateral surfaces of the frame 22 and lateral surfaces of the substrate21) are fixed to the placement surface 1 a of the sample stage 1 by anadhesive tape T having conductivity such as a carbon tape or the like.In this way, as the sample support 2 is fixed to the sample stage 1, thesample 10 and the sample support 2 are brought into close contact witheach other, and the movement of the sample 10 caused by the capillaryphenomenon (to be described below) can be more smoothly performed.Sideslippage of the sample support 2 arranged on the sample 10 can beprevented, and a loss of positional information of the sample 10 due tothe sideslip of the sample support 2 can be suppressed.

Here, when the sample stage 1 has conductivity, the sample stage 1 andthe sample support 2 are electrically connected by the adhesive tape Thaving conductivity. Therefore, a predetermined current is applied tothe sample stage 1 in the state in which the sample support 2 is fixedto the sample stage 1 by the adhesive tape T as illustrated in (a) ofFIG. 8, and thereby a predetermined voltage is applied to the substrate21. Thereby, a constant potential difference can be generated betweenthe aforementioned ground electrode and the substrate 21. In the presentembodiment, since the conductive layer 23 covers the frame 22 and theadhesive tape T is in contact with the conductive layer 23 on the frame22, the sample support 2 and a power source (a predetermined powersource that applies the current to the sample stage 1) can be broughtinto contact with each other on the frame 22. That is, the samplesupport 2 and the power source can be brought into contact with eachother without reducing the effective region R on the substrate 21.

As illustrated in (b) of FIG. 8, as described above, the sample support2 is arranged on the sample 10, and thereby the sample 10 is moved(raised) from the other surface 21 b side of the substrate 21 toward theone surface 21 a side via the through-holes S by the capillaryphenomenon. The sample 10 enters a state in which it stays on the onesurface 21 a side of the sample support 2 due to surface tension. Here,the placement surface 1 a of the sample stage 1 and the one surface 21 aand the other surface 21 b of the substrate 21 are arranged to be nearlyparallel to each other. Therefore, the sample 10 placed on the samplestage 1 is moved from the other surface 21 b side to the one surface 21a side of the substrate 21 via the through-holes S in a directionperpendicular to the placement surface 1 a of the sample stage 1 due tothe capillary phenomenon. Thereby, before and after the movement causedby the capillary phenomenon, the positional information of the sample 10(each sample molecule composing the sample 10) is maintained. In otherwords, two-dimensional coordinates (positions on a two-dimensional planeparallel to the placement surface 1 a of the sample stage 1) of eachsample molecule composing the sample 10 are not greatly changed beforeand after the movement caused by the capillary phenomenon. Accordingly,due to this capillary phenomenon, the sample 10 can be moved from theother surface 21 b side to the one surface 21 a side of the substrate 21while the positional information of the sample 10 is maintained.

Subsequently, as illustrated in FIG. 9, the laser beam L is applied tothe one surface 21 a of the substrate 21 by the laser beam applicationunit 4, and the sample 10 moved from the other surface 21 b side to theone surface 21 a side via the through-holes S by the capillaryphenomenon is ionized (a third process). The ionized sample 10 (thesample ions 11) is detected by the detector 3 (a fourth process). Whilechanging application positions of the laser beam L, the application ofthe laser beam L in the third process and the detection of the sampleions 11 in the fourth process are performed at each applicationposition. To be specific, the laser beam application unit 4 scans theeffective region R according to a predetermined movement width and apredetermined moving direction, and applies the laser beam L at eachapplication position while changing the application positions of thelaser beam L. The detector 3 detects the sample ions 11 emitted into avacuum by applying the laser beam L from the laser beam application unit4 at each application position. As a result, imaging mass spectrometryfor imaging a two-dimensional distribution map of sample molecules canbe performed on the basis of measurement results of the sample ions 11detected at each application position.

According to the SALDI method (the first to third processes), thesubstrate 21 in which the plurality of through-holes S are provided isarranged on the sample 10, and thereby the sample 10 can be raised fromthe other surface 21 b side toward the one surface 21 a side of thesubstrate 21 via the through-holes S due to a capillary phenomenon.Thereby, the sample 10 can be moved from the other surface 21 b side tothe one surface 21 a side of the substrate 21 while the positionalinformation of the sample 10 (the two-dimensional distribution of themolecules composing the sample 10) is maintained. The laser beam L isapplied to the one surface 21 a of the substrate 21, and energy istransmitted to the sample 10 moved to the one surface 21 a side via theconductive layer 23. Thereby, the sample 10 is ionized. As a result, thesample 10 can be ionized while the positional information of the sample10 is maintained. Therefore, according to the aforementioned method, thesample 10 can be ionized by a simple operation in which the substrate21, in which the plurality of through-holes S are provided, is placed onthe sample 10 while the positional information of the sample 10 ismaintained.

Al is anodized, and thereby the sample support 2 having the substrate 21in which the plurality of through-holes S are provided is used. Thereby,the movement of the sample 10 caused by the aforementioned capillaryphenomenon can be properly realized. Here, even when the sample support2 having the substrate 21 obtained by anodizing a valve metal other thanAl or Si instead of Al is used, the same effects are obtained.

The substrate 21 having the through-holes S, each of which has the holewidth d3 of 1 to 700 nm, is used, and thereby the movement of the sample10 caused by the aforementioned capillary phenomenon can be moresmoothly performed.

Since the sample support 2 has the frame 22 mounted on the outer edge ofthe one surface 21 a of the substrate 21, the bending of the substrate21 is suppressed by the frame 22, and the sample support 2 is easilyhandled when supported or moved. For this reason, the arrangement of thesample support 2 on the sample 10 in the second process can be easilyperformed.

According to the mass spectrometry method (the first to fourthprocesses), the sample 10 can be ionized by a simple operation in whichthe sample support 2 is arranged on the sample 10 while the positionalinformation of the sample 10 is maintained. While changing theapplication positions of the laser beam L, the ionized sample 10 (thesample ions 11) is detected at each application position, and therebythe two-dimensional distribution of the sample molecules can beperceived. Therefore, according to the mass spectrometry method, theimaging mass spectrometry for imaging the two-dimensional distributionmap of the sample molecules can be performed by the simple operation.

According to the mass spectrometry device 100, the sample 10 can beionized by a simple operation in which the sample support 2 is arrangedon the sample 10 while the positional information of the sample 10 ismaintained. The laser beam application unit 4 applies the laser beam Lwhile changing the application positions, and the detector 3 detects theionized sample 10 (the sample ions 11) at each application position, andthereby the two-dimensional distribution of the sample molecules can beperceived. Therefore, according to the mass spectrometry device 100,imaging mass spectrometry for imaging a two-dimensional distribution mapof sample molecules can be performed by the simple operation.

While the embodiment of the present invention has been described, thepresent invention is not limited to the embodiment and can be modifiedin various ways without departing from the gist thereof.

For example, the substrate 21 may be formed of a conductive materialsuch as a semiconductor. In this case, the sample support 2 can omit theconductive layer 23 for giving conductivity to the substrate 21. Whenthe sample support 2 is not provided with the conductive layer 23, thesample support 2 is arranged on the sample 10 such that the othersurface 21 b comes into direct contact with the sample 10 in the secondprocess. Even when the substrate 21 is formed of a conductive materialin this way and the sample support 2 from which the conductive layer 23is omitted is used, the same effects as when the sample support 2 havingthe aforementioned conductive layer 23 is used can be obtained.

The ionization of the sample 10 caused by the SALDI method (the first tothird processes) can also be used for other measurements and experimentssuch as ion mobility measurement as well as the imaging massspectrometry of the sample 10 which is described in the presentembodiment.

The conductive layer 23 may be provided by vapor deposition or the liketo cover at least the one surface 21 a of the substrate 21. That is, theconductive layer 23 may not be provided on the other surface 21 b of thesubstrate 21 and the inner surfaces of the through-holes S. In thiscase, the sample support 2 is arranged on the sample 10 such that theother surface 21 b faces the sample 10 in the second process, and theother surface 21 b comes into direct contact with the sample 10. If theconductive layer 23 is provided to cover the surface of the frame 22 andat least the one surface 21 a of the substrate 21, the contact betweenthe substrate 21 and the electrode can be made on the frame 22.

FIGS. 10 to 13 illustrate a relation between the hole width of each ofthe through-holes S and a mass spectrum measured by the massspectrometry method. Here, as the sample support, a sample support inwhich the conductive layer 23 (here, Pt) is provided to cover the onesurface 21 a and the surface of the frame 22 without being provided onthe other surface 21 b of the substrate 21 and the inner surfaces of thethrough-holes S is used. The thickness d1 of the substrate 21 is 10 μm,and the sample to be measured is a peptide of “mass-to-charge ratio(m/z)=1049.” In FIGS. 10 to 13, (a) shows measured results when the holewidths of the through-holes S are set to 50 nm, (b) shows measuredresults when the hole widths of the through-holes S are set to 100 nm,(c) shows measured results when the hole widths of the through-holes Sare set to 200 nm, (d) shows measured results when the hole widths ofthe through-holes S are set to 300 nm, (e) shows measured results whenthe hole widths of the through-holes S are set to 400 nm, (f) showsmeasured results when the hole widths of the through-holes S are set to500 nm, (g) shows measured results when the hole widths of thethrough-holes S are set to 600 nm, and (h) shows measured results whenthe hole widths of the through-holes S are set to 700 nm. In FIGS. 10 to13, the longitudinal axis denotes signal intensity (Intensity)standardized as 100(%) for a peak value.

As illustrated in FIGS. 10 to 13, even when the hole widths of thethrough-holes S of the substrate 21 are any one of 50 nm, 100 nm, 200nm, 300 nm, 400 nm, 500 nm, 600 nm, and 700 nm, a proper spectrum inwhich a peak can be observed is obtained. In this way, the samplesupport having the substrate 21 in which the conductive layer 23 isprovided on at least the one surface 21 a is used, and thereby the massspectrometry can be properly performed.

FIG. 14 illustrates a relation between the thickness d1 (Thickness) ofthe substrate 21 and signal intensity of a peak measured by the massspectrometry method. In FIG. 14, the longitudinal axis denotes relativesignal intensity (Relative intensity) of the case the signal intensityis set to “1” when the thickness d1 of the substrate 21 is 10 μm. Here,as the sample support, a sample support in which the conductive layer 23(here, Pt) is provided to cover the one surface 21 a and the surface ofthe frame 22 without being provided on the other surface 21 b of thesubstrate 21 and the inner surfaces of the through-holes S is used. Thehole widths of the through-holes S are 200 nm. The sample to be measuredis a peptide of “mass-to-charge ratio (m/z)=1049”.

In the measured results, the signal intensity when the thickness d1 ofthe substrate 21 is 10 μm is of sufficient magnitude for massspectrometry. As illustrated in FIG. 14, as the thickness d1 of thesubstrate 21 becomes smaller, the signal intensity shows a tendency toincrease. When the thickness d1 of the substrate 21 ranges from 3 to 10μm, sufficient signal intensity is obtained. Meanwhile, in terms ofsecuring strength of the substrate, the thickness d1 of the substrate 21is preferably large. For this reason, the thickness d1 of the substrate21 may be set to 5 to 10 μm. Thereby, the strength of the substrate 21can be maintained, and sufficient signal intensity can be obtained inthe mass spectrometry.

In the embodiment, the form in which the frame 22 of the sample support2 is fixed to the sample stage 1 by the adhesive tape T has beendescribed, but a form of fixing the sample support 2 to the sample stage1 is not limited to the form. Hereinafter, a variation of the form offixing the sample support 2 to the sample stage 1 will be describedusing FIGS. 15 to 17 along with first to third modifications of thesample support 2. In FIGS. 15 to 17, the conductive layer 23 and thethrough-holes S are not illustrated. In FIGS. 16 and 17, the bondinglayer G for bonding the frame and the substrate is also not illustrated.

(First Modification)

As illustrated in FIG. 15, a sample support 2A according to a firstmodification is mainly different from the sample support 2 in that aframe 22 is not provided for a substrate 21 and an adhesive tape T isdirectly stuck on one surface 21 a of the substrate 21. The adhesivetape T is stuck on an outer edge of the one surface 21 a such that anadhesive face Ta thereof faces the one surface 21 a of the substrate 21,and the adhesive tape T has a portion that extends beyond an outer edgeof the substrate 21. Thereby, as illustrated in FIG. 15, the adhesiveface Ta can be stuck on the outer edge of the substrate 21 and aplacement surface 1 a of a sample stage 1. As a result, the samplesupport 2A is fixed to the sample stage 1 by the adhesive tape T.According to the sample support 2A, for example when mass spectrometryof a sample 10 whose surface has concavities and convexities isperformed, a follow-up characteristic of the substrate 21 for the sample10 can be improved.

When the sample stage 1 has conductivity, the sample stage 1 and thesample support 2A (particularly, a conductive layer 23 provided on theone surface 21 a of the substrate 21) are electrically connected via theadhesive tape T having conductivity. Therefore, as illustrated in FIG.15, in the state in which the sample support 2 is fixed to the samplestage 1 via the adhesive tape T, a predetermined current is applied tothe sample stage 1, and thereby a predetermined voltage can be appliedto the substrate 21.

The sample support 2A may be distributed in a state in which theadhesive tape T is stuck on the outer edge of the substrate 21 and anadhesive protection sheet is provided on the adhesive face Ta of theportion that extends beyond the outer edge of the substrate 21. In thiscase, a user of the sample support 2A releases the adhesive protectionsheet immediately before the sample support 2A is fixed to the samplestage 1, and sticks the adhesive face Ta on the placement surface 1 a,and thereby preparation of the mass spectrometry of the sample 10 can beeasily performed.

(Second Modification)

As illustrated in FIG. 16, a sample support 2B according to a secondmodification is mainly different from the sample support 2 in that aframe 122 having a portion that extends beyond an outer edge of asubstrate 21 is provided. When the sample support 2B is carried by thisframe 122, damage to an end of the substrate 21 can be properlysuppressed. Further, as illustrated in FIG. 16, insertion holes 122 afor inserting screws 30 are provided in the portion of the frame 122which extends beyond the outer edge of the substrate 21. In this case,for example when a sample stage 1A having screw holes 1 b at positionscorresponding to the insertion holes 122 a is used, the sample support2B can be reliably fixed to the sample stage 1A by screwing. To bespecific, the screws 30 are inserted into the insertion holes 122 a andthe screw holes 1 b, and thereby the sample support 2B can be fixed tothe sample stage 1A.

When the sample stage 1A has conductivity and when the screws 30 haveconductivity, the sample stage 1A and the sample support 2B(particularly, a conductive layer 23 formed on the frame 122) areelectrically connected via the screws 30. Therefore, as illustrated inFIG. 16, in a state in which the sample support 2B is fixed to thesample stage 1A via the screws 30, a predetermined current is applied tothe sample stage 1A, and thereby a predetermined voltage can be appliedto the substrate 21.

(Third Modification)

As illustrated in FIG. 17, a sample support 2C according to a thirdmodification is mainly different from the sample support 2 in that anadhesion layer 24 having one adhesive face 24 a facing a directiondirected from one surface 21 a to the other surface 21 b is provided atan outer edge of the other surface 21 b of a substrate 21. The adhesionlayer 24 is, for instance, a double-sided tape or the like that has athickness predetermined depending on a thickness of a sample 10 to bemeasured. For example, the other adhesive face 24 b of the adhesionlayer 24 is previously stuck on the outer edge of the other surface 21 bof the substrate 21, and the one adhesive face 24 a of the adhesionlayer 24 is stuck on a placement surface 1 a when the sample support 2Cis fixed to a sample stage 1. According to the sample support 2C, aconfiguration in which the sample support 2C is fixed to the samplestage 1 can be simplified.

When the sample stage 1 has conductivity and when the adhesion layer 24has conductivity, the sample stage 1 and the sample support 2C(particularly, the substrate 21) are electrically connected via theadhesion layer 24. Therefore, as illustrated in FIG. 17, in a state inwhich the sample support 2C is fixed to the sample stage 1 via theadhesion layer 24, a predetermined current is applied to the samplestage 1, and thereby a predetermined voltage can be applied to thesubstrate 21.

The sample support 2C may be distributed in a state in which theadhesive face 24 b of the adhesion layer 24 is stuck on the outer edgeof the other surface 21 b of the substrate 21 and an adhesive protectionsheet is provided for the adhesive face 24 a. In this case, a user ofthe sample support 2C releases the adhesive protection sheet immediatelybefore the sample support 2C is fixed to the sample stage 1, and sticksthe adhesive face 24 a on the placement surface 1 a, and therebypreparation of the mass spectrometry of the sample 10 can be easilyperformed.

The sample supports 2, 2A, 2B, and 2C according to the embodiment andthe modifications may be baked after the conductive layer 23 is formed.The process of manufacturing a sample support in the embodiment mayinclude a baking process of baking the sample support after theconductive layer 23 is formed. When the frame 22 is provided, the bakingprocess is performed on a sample support having the substrate 21, theframe 22, and the conductive layer 23. When the frame 22 is omitted, thebaking process is performed on a sample support having the substrate 21and the conductive layer 23.

By performing this baking process, crystallinity of the conductive layer23 (for instance, Pt) can be improved, and a sample support that is moresuitable for mass spectrometry can be obtained. Here, the baking of thesample support is preferably performed such that a diffraction peak of acrystal of a conductive material (here, Pt) forming the conductive layer23 is shown in an X-ray diffraction (XRD) measurement for the conductivelayer 23 (the sample support) after the baking. Here, the expression ofthe “diffraction peak of the crystal of the conductive material isshown” means that a diffraction pattern (peak intensity or the like) ofthe crystal of the conductive material is more clearly shown thanmeasured results obtained by the XRD measurement for the sample supportbefore the baking.

(a) of FIG. 18 illustrates a mass spectrum measured by the massspectrometry device 100 having the sample support before being baked. Onthe other hand, (b) of FIG. 18 illustrates a mass spectrum measured bythe mass spectrometry device 100 having the sample support after beingbaked at a baking temperature of 400° C. Measurement conditions (a typeof sample, a configuration of the sample support, etc.) other than thepresence and absence of the baking are the same between (a) and (b) ofFIG. 18. The longitudinal axes of (a) and (b) of FIG. 18 denote relativesignal intensity of the case signal intensity of a peak (that is, a peakvalue of the graph in (b) of FIG. 18) is set to “100” when the samplesupport after the baking is used. As illustrated in FIG. 18, the signalintensity can be further improved in mass spectrometry by using thesample support after the baking than when using the sample supportbefore the baking. In this way, a sample support that is more suitablefor the mass spectrometry can be obtained by performing the bakingprocess.

REFERENCE SIGNS LIST

1 Sample stage

2, 2A, 2B, 2C Sample support

3 Detector

4 Laser beam application unit

10 Sample

11 Sample ion

21 Substrate

21 a One surface

21 b Other surface

22, 122 Frame

23 Conductive layer

24 Adhesion layer

24 a, 24 b Adhesive face

30 Screw

122 a Insertion hole

L Laser beam

S Through-hole

T Adhesive tape

Ta Adhesive face

The invention claimed is:
 1. A surface-assisted laser desorption/ionization method comprising: a first process of preparing a sample support having a substrate in which a plurality of through-holes passing from one surface thereof to the other surface thereof are provided and a conductive layer that is formed of a conductive material and covers at least a portion of the one surface not provided with the through-holes so that each opening of the through-holes is not covered by the conductive layer; a second process of placing a sample on a sample stage and arranging the sample support on the sample such that the other surface faces the sample; and a third process of applying a laser beam to the one surface and ionizing the sample moved from the other surface side to the one surface side via the through-holes due to a capillary phenomenon.
 2. The surface-assisted laser desorption/ionization method according to claim 1, wherein the substrate is formed by anodizing a valve metal or silicon.
 3. The surface-assisted laser desorption/ionization method according to claim 1, wherein each of the through-holes has a width of 1 to 700 nm.
 4. The surface-assisted laser desorption/ionization method according to claim 1, wherein the substrate has a thickness of 5 to 10 μm.
 5. The surface-assisted laser desorption/ionization method according to claim 1, wherein the sample support further includes a frame mounted on an outer edge of the one surface of the substrate.
 6. The surface-assisted laser desorption/ionization method according to claim 1, wherein in the second process, the sample support is fixed to the sample stage.
 7. A surface-assisted laser desorption/ionization method comprising: a first process of preparing a sample support having a substrate which is formed of a conductive material and in which a plurality of through-holes passing from one surface thereof to the other surface thereof are provided; a second process of placing a sample on a sample stage and arranging the sample support on the sample such that the other surface is in contact with the sample; and a third process of applying a laser beam to the one surface and ionizing the sample moved from the other surface side to the one surface side via the through-holes due to a capillary phenomenon.
 8. A mass spectrometry method comprising: each of the processes of the surface-assisted laser desorption/ionization method according to claim 1; and a fourth process of detecting the sample ionized in the third process, wherein the application of the laser beam in the third process and the detection of the ionized sample in the fourth process are performed at each application position while changing application positions of the laser beam. 